Polymorphonuclear leukocytes (leukocytes) provide a principle means of defense against microbial infections. The response to invading microorganisms causes activation of cellular oxidative processes (production of hydroxyl radicals) and nonoxidative processes (digestive enzymes; myeloperoxidase, elastase, etc.) in order to effectively kill the microorganisms. However, the response of leukocytes to a foreign challenge can also cause destruction to host tissues and play an important part in the pathogenesis of a number of noninfectious disease conditions.
Leukocytes possess a wide variety of mechanisms that enable them to respond to foreign challenges which are initiated by cell surface receptors. Receptor activation or general cellular activation results in an altered cellular physiology causing the cell in itself to become "activated". The intracellular signaling molecules of activation are often referred to as second messengers, the first messengers being the extracellar activating ligands themselves.
One of the major second messengers in many cells is the calcium ion (Ca.sup.+2). There are two general ways in which cell-surface receptors are known to generate intracellular calcium signals. One is by activating phospholipase C. This enzyme generates inositol trisphosphate which, in turn, releases stored calcium in the cell. Alternatively, cell receptors may open or close gated ion channels, letting calcium enter from outside the cell. Ca.sup.+2 channels in the plasma membrane are of two types: (1) voltage sensitive calcium channels which are activated when a small and transient flux of ions briefly alter the voltage across the plasma membrane, or (2) receptor operated channels which are directly opened by receptor ligands. The first mechanism operates mainly in voltage sensitive cells such as neurons and muscle cells. Many cells, like leukocytes, are not primarily voltage sensitive cells but have cell-surface receptors that are functionally linked to receptor sensitive Ca.sup.+2 channels in the plasma membrane. Binding of certain ligands activates these receptors, thereby opening the channels and allowing Ca.sup.+2 to enter the cytosol, where it then functions as a second messenger.
When cells are activated, corresponding to an influx of Ca.sup.+2, structures within the cell that bind Ca.sup.+2 are responsive to such changes, depending on their relative affinity and specificity for calcium. A few Ca.sup.+2 dependent proteins are known. The first such protein to be discovered and characterized was troponin C found in electrically active skeletal muscle cells. A later discovered calcium binding protein which is ubiquitous in both voltage and receptor sensitive cells is calmodulin. Among the increasing number of cellular proteins known to be regulated by calmodulin in a Ca.sup.+2 dependent manner are some forms of cyclic nucleotide phosphodiesterase and adenylate cyclase, as well as membrane bound calcium dependent ATPases, phosphorylase kinase, myosin light chain kinases, and their association with the spindles of the mitotic apparatus and the bundles of actin filaments. Although the total number of proteins that are calcium dependent or are affected by Ca.sup.+2 dependent enzymes is not known it is clear that calcium is a requirement as a means of activating these processes.
When leukocytes are activated, a number of events can occur which are important in leading to intracellular calcium mediated disease states. For example leukocytes, primarily the neutrophils, are thought to play an integral part in the symptoms and tissue injury of the host in the following diseases; gout, rheumatoid arthritis, immune vasculitis, glomerulonephritis, inflammatory bowel disease, adult respiratory distress syndrome, emphysema, asthma, thermal injury associated with hemolysis, and malignant neoplasms at sites of chronic inflammation (Malech and Gallin; 1987). It therefore appears desirable to inhibit Ca.sup.+2 uptake in leukocytes in order to alleviate or slow the progression of these immune and inflammatory diseases associated with calcium uptake. Ca.sup.+2 uptake in leukocytes and amelioration of immune and inflammatory diseases may also be extended to those diseases associated with the skin and dermal tissues. Included in the list of those topically related inflammatory diseases are those associated with skin and dermis, including neutrophil dermatoses, chronic dermatitis, psoriasis, contact dermatitis, atopic and seborrheic dermatitis, and acne.
Calcium is also an important mediator in thrombocytes (platelets) where it is well known that Ca.sup.+2 is a required mediator in the intrinsic pathway of blood coagulation. For example, the Ca.sup.+2 requirement in the blood clotting process and thrombus formation is well known and these processes can be inhibited in vitro, and to some degree in vivo, if chelating agents such as EDTA, citrate, or oxalate are added to bind Ca.sup.+2. It is recognized that Ca.sup.+2 plays an integral part of the fibrinolytic cascade and is an active mechanism by which the fibrinolytic response can be modulated therapeutically.
One of the primary functions of platelets occurs at a site of vascular injury wherein they form clot aggregates to close the wound. This response can also have a number of detrimental side effects, for example during ischemic reperfusion the thrombus formation can lead to occlusion of the artery, to the extent of causing a myocardial infarction. It is therefore desirable in many instances to inhibit Ca.sup.+2 uptake in platelets in order to control the thrombolytic response.
Ca.sup.+2 entry into the cytosol, by various forms of receptor-mediated activation or by discharge of intracellular stores, is critically linked to certain cellular events of leukocyte activation and platelet aggregation. Altering pathways of Ca.sup.+2 mobilization provides a mechanism to modulate the responses of leukocytes and platelets. Therefore compounds that inhibit Ca.sup.+2 mobilization might be expected to reduce Ca.sup.+2 dependent disease processes associated with leukocyte activation and inhibition in immune and inflammatory diseases and in problems involving platelet aggregation in certain thrombolytic conditions.